1. Field of the Invention
The present invention relates to separation matrices useful for removing microorganism-originated contaminants from biological liquids and methods for their preparation and use. The separation matrices may be used as chromatographic separation media as well as providing the added benefit of antimicrobial activity and are characterized by the presence of immobilized polyionene.
2. Brief Description of the Background Art
The broad applicability of ion exchange chromatography, which ranges from separation of inorganic and organic ions to that of protein molecules and other biomolecules, has made it a powerful and versatile tool for chemical and biochemical separations. The technique was originally limited to the use of natural products such as cellulose, clay and other minerals containing mobile ions that would exchange with ionic materials in the surrounding solute phase. Because of the low exchange capacity of these natural products, however, practical utilization thereof was limited, and synthetic organic polymers capable of exchanging ions were developed.
Among the first generation of synthetic ion exchange materials were the ion exchange resins. The fundamental framework of these ion exchange resins is an elastic three-dimensional hydrocarbon network comprising ionizable groups, either cationic or anionic, chemically bonded to the backbone of a hydrocarbon framework. The network is normally fixed, insoluble in common solvents and is chemically inert. The ionizable functional groups attached to the matrix carry active ions which can react with or can be replaced by ions in the solute phase. Therefore, the ions in the solute phase can be easily exchanged for the ions initially bound to the polymeric resins. Typical examples of commercially available ion exchange resins are the polystyrenes cross-linked with DVB (divinylbenzene), and the methacrylates copolymerized with DVB. In the case of polystyrene, a three-dimensional network is formed first, and the functional groups are then introduced into benzene rings through chloromethylation. Since ion exchange resins are elastic three-dimensional polymers, they have no definite pore size; only a steadily increasing resistance to flow of the polymer network limits the uptake of ions and molecules of increasing size.
The resistance to flow exhibited by these resins is controlled by the degree of crosslinking. With a low degree of crosslinking, the hydrocarbon network is more easily stretched, the swelling is large, and the resin exchanges small ions rapidly and even permits relatively large ions to undergo reaction Conversely, as the crosslinking is increased, the hydrocarbon matrix is less resilient, the pores of the resin network are narrowed, the exchange process is slower, and the exchanger increases its tendency to exclude large ions from entering the structure. The ion exchange resins made from polymeric resins have been successfully applied for the removal of both organic and inorganic ions from aqueous media but they are normally unsuitable for the separation of biopolymers such as proteins. This is due, among others, to the following reasons:
(1) The highly crosslinked structure of the resins has rather narrow pores to accommodate the diffusion of proteins; the proteins therefore are virtually restricted to the macrosurface area of the beads with consequent limitation of solute loadings;
(2) The high charge density close to the proximity of the resin surface is unsuitable, since it causes excessive binding and distortion of protein structure;
(3) The hydrocarbon matrix is usually hydrophobic and is potentially dangerous to the subtle three-dimensional structure of biopolymers, often causing denaturation of proteins.
The next generation of chromatographic materials useful for separation of proteins and other labile biological substances was based on cellulose ion exchangers. These lacked nonspecific adsorption and had practicable pore structure. Such prior art ion exchange celluloses are made by attaching substituent groups with either basic or acidic properties to the cellulose molecule by esterification, etherification, or oxidation reactions. Examples of cationic exchange celluloses are carboxymethylated cellulose (CM), succinic half esters of cellulose, sulfoethylated cellulose, and phosphorylated cellulose. Examples of anionic exchange celluloses are diethylaminoethyl cellulose (DEAE), and triethylaminoethyl cellulose (TEAE). Ion exchange materials based on cellulose as the principal backbone or anchoring polymer, however, have not enjoyed complete success due primarily to an inherent property of cellulose: its affinity for water. Thus, prior art ion exchange materials based on cellulose, while typically having high exchange capacity, are difficult to use as a consequence of their tendency to swell, gelatinize or disperse on contact with an aqueous solution. An ideal ion exchange material should minimally interact with the solvent system which carries the ions in solution through its pores; only in this manner is it possible to obtain a rapid, free flowing ion exchange system.
A third generation of ion exchange materials, which were developed to solve some of these problems, were the ion exchange gels. There gels comprise large pore gel structures and include the commercially known material Sephadex.RTM., which is a modified dextran. The dextran chains are crosslinked to give a three-dimensional polymeric network. The functional groups are attached by ether linkages to the glucose units of the dextran chains. Proteins are not denatured by the hydrophilic polymeric network. Sephadex.RTM. exhibits very low nonspecific adsorption, which makes it ideal as a matrix for biological separations. However, the porosity of ion exchange gels is critically dependent on its swelling properties, which in turn is affected by the environmental ionic strength, pH and the nature of the counter-ions. Swelling of gels in buffer is caused primarily by the tendency of the functional groups to become hydrated. The amount of swelling is directly proportional to the number of hydrophilic functional groups attached to the gel matrix, and is inversely proportional to the degree of crosslinking present in the gel. This characteristic swelling is a reversible process, and at equilibrium there is a balance between two forces: (1) the tendency of the gel to undergo further hydration, and hence to increase the osmotic pressure within the gel beads, and (2) the elastic forces of the gel matrix. The osmotic pressure is attributed almost entirely to the hydration of the functional groups, and, since different ions have different degrees of hydration, the particular counter-ions in an ion exchange gel can be expected to have a considerable influence upon the degree of swelling. Since the pH, the electrolyte concentration and the nature of the counter-ions can all affect the hydration, leading to a different degree of gel swelling, the pore size in the gels is not in well defined form but is rather dependent on the environmental conditions. Gels without crosslinking provide large pores and high capacity due to maximum swelling. They suffer, however, from the weakness of structural integrity and can easily be crushed with a minimum amount of pressure. Removal of the solvent from the gels often results in collapse of the matrix. Highly crosslinked gels have mechanical strength, but lose capacity and pore size due to restrictions in swelling.
Ion exchange gels made from synthetic polymers have also been used, and they include crosslinked polyacrylamide (Bio-Gel P.RTM.), microreticular forms of polystyrene (Styragel.RTM.), poly(vinyl acetate) (Merck-o-Gel OR.RTM.), crosslinked poly(2-hydroxy ethylmethacrylate)(Spheron.RTM.), and polyacryloylmorpholine (Enzacryl.RTM.). All of these follow the general trend: it may be possible to obtain dimensional stability with high flow rate or, alternatively, high capacity with swelling. It is, however, not possible to obtain both capacity and high flow rate at the same time.
The failure of single components to have both capacity and dimensional stability led to yet another generation of ion exchange materials comprising composite structures, e.g., hybrid gels. Hybrid gels are made by combining a semi-rigid component, for the purpose of conferring mechanical stability, with a second component, a softer network, which is responsible for carrying functional groups. Agarose gel, which would otherwise be very soft and compressible, can be made stronger through hybridizing with crosslinked polyacrylamide. The crosslinked polyacrylamide component is mechanically stronger than the agarose, improves the gel flow properties, and reduces the gel swelling, but it sacrifices molecular fractionation range. Examples of hybrid gels other than polyacrylamide/agarose (Ultrogels.RTM.), are polyacryloylmorpholine and agarose (Enzacryl.RTM.), and composite polystyrenes with large pore polystyrenes as a framework filled with a second type of lightly crosslinked polymer.
Yet another composite gel structure is achieved by combining inorganic materials coated with organics, and are the types known as Spherosil.RTM.. Porous silica beads are impregnated with DEAE dextran so that the product will have the mechanical properties of silica, with the ion exchange properties of DEAE dextrans. These composites, however, have severe channeling defects arising out of particle packing, and they have capacity limitations on the coated surfaces.
Totally rigid inorganic supports such as porous silica or porous glass which are not susceptible to degradation have also been used to provide high porosity, and high flow rate systems. The major problem, however, is nonspecific adsorption of proteins due to the silanol groups on the silica surface. Since the hydrolysis of silica is directly related to the pH conditions, the nonspecific adsorption by silica is minimal at neutral pH, but increases as the pH changes both to the acidic or alkaline ranges. A monolayer coating by either hydrophilic organic polymers or silanization has been used in an attempt to overcome this problem.
In the technique of affinity chromatography, which enables the efficient isolation of biological macromolecules or biopolymers, by utilizing their recognition sites for certain supported chemical structures with a high degree of selectivity, the prior art has also utilized materials of varying chemical structure as supports. For example, agarose gels and crosslinked agarose gels have been the most widely used support materials. Their hydrophilicity makes them relatively free of nonspecific binding, but their compressibility makes them less attractive as carriers in large scale processing, such as in manufacturing. Controlled-pore glass (CPG) beads have also been used in affinity chromatography. Although high throughputs can be obtained with columns packed with CPG, this carrier is even more expensive than agarose gel beads. Cellulose particles have also been used by immunochemists for synthetic affinity sorbents. However, compared to agarose gels, cellulose particles are formed with more difficulty and therefore, have received less attention in the preparation of affinity sorbents for enzymes. Cellulose, however, is perhaps the least expensive of all support matrices. Two lesser used support matrices are polyacrylamide gel beads and Sephadex.RTM. gel beads made from dextran and epichlorohydrin. Although convenient methods have been developed for using them, the softness of these beads yields poor column packings, and their low molecular porosity yields a sorbent with poor ligand availability to the ligate.
Coupek et al., U.S. Pat. No. 4,281,233 show supports for affinity chromatography which comprise copolymers of hydroxy alkyl acrylates or methacrylates with crosslinking monomers. The copolymers contain covalently attached mono- or oligosaccharides. (An oligosaccharide is defined in the art as having up to nine saccharide units. See, e.g., Roberts, J. D., and Caserio, M. C., Basic Principles of Organic Chemistry, 1964, p. 615.)
A carrier for bio-active materials is also disclosed in Nakashima et al., U.S. Pat. No. 4,352,884. The Nakashima carrier comprises a substrate coated with a copolymer. The substrate may be one of various materials, including inorganic materials such as glass, silica, alumina, synthetic high polymers such as polystyrene, polyethylene and the like, and naturally occurring high polymers such as cellulose. The copolymer is made of a hydrophilic acrylate or methacrylate monomer which is a hydroxy or alkoxy alkyl acrylate or methacrylate, and a copolymerizable unsaturated carboxylic acid or amine. The base material or substrate is coated with the copolymer by conventional coating or deposition procedures, such as spraying, dipping, phase separation or the like. The copolymer may also contain small amounts of a crosslinking agent such as glycidyl acrylate or methacrylate. The crosslinking agent allows for cross-linking treatment after the coating process, and provides for the prevention of elution (presumably of the bioactive materials) from the coating layer. The amounts of cross-linking agent are quite small, and range between 0.5 and 1 percent by weight of the total copolymer weight. Such amounts of cross-linking agent are insufficient to cause substantial covalent bonding or grafting of the copolymer onto the underlying substrate. The copolymer in Nakashima is thus essentially only physically coating the underlying substrate. Physical coating, however, is accompanied by a series of problems. The carrier would not be expected to have an even distribution of the copolymer, would show a multilayered structure, and may have a possible uneven distribution of functional groups.
Another reference of interest is Kraemer, U.S. Pat. No. 4,070,348, which shows copolymers of glycidyl- and amino-containing acrylates, useful as carriers for biologically active substances, such as polysaccharides, enzymes, peptides, hormones, etc. The structure of the final product in Kraemer is that of an acrylic copolymer chain covalently modified at a multiplicity of sites thereon with substances such as enzymes, proteins, and the like.
This review of the prior art, its advantages and drawbacks, leads to the conclusion that there exists a need for a support useful both for ion exchange and affinity chromatography-based purification which will have high stability, high porosity, low nonspecific adsorption, high flow rate, poor compressibility, controlled gelation, and which will be useful for industrial-scale biological separations. It is at the industrial level of manufacturing, especially, where the aforementioned drawbacks have had their most important effect and where this need is the strongest.
Industrial scale molecular separation materials comprising fibrous matrices with particulate immobilized therein have been described in commonly assigned U.S. Pat. No. 4,384,957 to Crowder, which is herein incorporated by reference. This patent describes a composite fiber material formed by wet layering a sheet from an aqueous slurry of particulate, small refined fiber pulp and long soft fiber pulp. The purpose of the soft long fiber is physically to hold clumps of particulate material and refined pulp together. Sheets are formed from a wet slurry by vacuum filtration, wherein the long fibers form in a plane which is perpendicular to the direction of flow of the chromatographic carrier fluid. This permits channels to form in the composite material which are perpendicular to the direction of flow of the chromatographic carrier fluid and allows these materials to serve as internal flow distributors. The resulting matrix structure has proven to be an effective way of eliminating channeling defects through internal flow distribution mechanisms.
It is inevitable in prior art wet slurrying processes with slurries comprising cationic materials, to obtain materials having uneven distribution of charges, wherein multilayer coating may occur in one spot, whereas other spots on the surface may be bare. Such products are acceptable in filtration processes due to the fact that the amount of impurities needed to be removed is relatively small compared to the bulk liquid volume, and that uneven charge distributions can be compensated by the depth of the filters. However, such products cannot readily be applied to delicate ion exchange processes. The number of active sites, as well as the accessibility of the active sites, is critical to the capacity of such process. The chemical functional groups in ion exchangers cannot be buried close to the surface, but have to be somewhat removed from the surface, possibly with a molecular side arm for accessibility. One way of achieving this has been through the incorporation into the fibrous matrix of silanes which are chemically modified. Such silanes may carry functional groups such as DEAE, CM or affinity chromatography sites. They are mechanically stable and strong and do not swell. However, they are expensive, and show very high nonspecific adsorption of protein by the silica hydroxy groups.
In sum, neither the ion exchange nor affinity chromatography supports commonly used in laboratory scale purifications, nor the particulate (or ion exchange modified particulate) containing fibrous matrices for chromatography or filtration have proven to be of great use in scale-up of delicate purification processes.
A need therefore continues to exist for supports useful in industrial scale ion exchange and affinity chromatography purification processes, which will be noncompressible, controllably swellable, have high exchange capacity, exhibit high flow rates, be versatile and be relatively inexpensive to produce. Recognizing this need, Hou et al. developed the invention embodied in the aforementioned application Ser. Nos. 466,114 and 576,448.
Purification of protein from bacteria contamination remains a recalcitrant problem in bioprocess. Hou et al, U.S. Pat. No. 4,361,486, discloses a bacteriocidal filter media which comprises an amount of metal peroxide immobilized in a substantially inert porous matrix. Both gram-positive and gram-negative bacteria are negatively charged, mainly owing to an excess of carboxyl and phosphate groups. Gram-positive bacteria contain both teichoic and teichuronic acids in their walls, whereas gram-negative organisms have phospholipids, with the negatively charged lipid portion of lipopolysaccharides as components of their outer membranes. In aqueous environments, the cell membrane exists as a continuum of lipid and protein, organized as a molecular double layer, with the hydrophobic portions of the lipid molecule being opposed and the hydrophilic groups projecting outwardly into the aqueous phase.
Phosphoglycerides account for about half the lipids, with polar groups, such as glycerol, serine, and carboxyl providing the hydrophilic components. While various forms of proteins are embedded in the lipid, the major determinants of charge are surface polysaccharides covalently linked to the membrane proteins and lipids.
The effectiveness of bacteria removal through charge interaction has been previously demonstrated, for example, by Ostreicher et al., U.S. Pat. Nos. 4,305,782 and 4,473,474 and Barnes, U.S. Pat. No. 4,473,475. Capture of bacteria, endotoxins, and viruses by charge modified filters are described in Applied and Environmental Microbiology, 40: 892-896 (1980). Positively charged ion exchange resins have been utilized for bacteria adsorption (Daniels, S. L., Development and Industrial Microbiology, 13: 211-253 (1972)).
Olson et al, U.S. Pat. No. 4,411,795 describes a variety of polymers attached to substrates including cellulose and recognized that the combined effect of hydrophobic and ionic binding enhances adsorption of lipnin-containing cells.
Zvaginstev, D. G. et al., Mikrobiologiya 40: 123-126 (1971), concluded that adsorption of bacterial cells by ion exchange resins was attributable to electrostatic attraction between quaternary ammonium groups on the resin surface and carboxyl groups on the bacteria cell surface. Hogg, in his Ph.D. thesis for the University of Salford, England (1976), demonstrated the interaction of bacteria with cellulose-based DEAE. The adsorption of several gram-negative organisms was shown, including Escherichia coli, Salmonella typhimurium, and Pseudomonas aeruginosa.
The major forces impacting on bacterial adhesion to solid surfaces have been summarized by Rutter, P. R. in "The Physical Chemistry of the Adhesive of Bacteria and Other Cells," (1980) Microbial Adhesion to Surfaces, Editors Berkley et al., Ellis Howard Ltd., Publishers, West Sussex, England. According to Rutter, the Van der Walls force and charge interaction may be considered as long range forces. Where the distance between bacteria and solid surfaces are short, other interactions must be taken into account, for example, ion-dipole, dipole-dipole, hydrogen bonding, etc. The short range effects are particularly important in aqueous systems. When the bacteria particles approach the microscopic solid surface, the local ordered water structure near the surface must be broken down. This leads to a short range repulsion force, which may be sufficient to prevent the bacteria from coming closer to the solid surface. On the other hand, when both the surfaces involved are hydrophobic, the short range interaction is a net attraction. This energy favorable process, called "hydrophobic interaction", is the basis for the well-known high performance liquid chromatography applied in protein separations. As is known, a hydrocarbon chain of optimum length, when attached covalently to a solid matrix, may adsorb one protein in preference to another due to the difference in hydrophobicity between proteins.
However, an overly strong hydrophobic solid surface may uncoil the protein structure leading to the exposure of hydrophobic regions and increase tendency for hydrophobic interaction. If the uncoiling is too extensive, denaturation of the protein may result.
In most of the practical applications for bacteria and endotoxin inactivation and removal from biological and pharmaceutical products, protein contamination by bacteria is the most prevalent problem. One must be able to inactivate and remove the microorganism-originated contaminants from protein specifically without causing loss or denaturation of the final products. Accordingly, an optimal solid matrix should exhibit a hydrophobic force which just matches the surface hydrophobicity of proteins and maximally exploits these selected differences in such hydrophobicity.
Thus, a need has continued to exist for a solid matrix for removal of microorganism-originated contaminants from biological and pharmaceutical products which will effectively eliminate the contaminants without denaturing the final product.
Poly-quaternary ammonium polymeric polyelectrolytes are known to the prior art, these polymeric compositions produced by the polymerization of a dihalide and a ditertiary amine. These polymers are characterized by high charge density and have found substantial utility as flocculants in the clarification of residential and industrial water supplies, as catalysts in pigment retention additives, and as geling agents. These polyeletrolyte materials are also known to be useful in the rheological modification of fluids such as friction reducers, as dispersants for clay and sludge in both aqueous and oil-based systems, as anti-static agents, and as additives to cosmetics, textile finishes and lubricating oils. The materials are known to exhibit germicidal action or effective bactericidal and fungicidal agents. See Rembaum et al., U.S. Pat. No. 3,898,188.
Buckman et al., U.S. Pat. No. 3,784,649, discloses "high molecular weight" ionene polymeric compositions for utility, among others, as broad spectrum microbicides for efficient control of bacteria including sulphate reducers, fungi, algae, and yeast. The Buckman et al. polyionenes are suggested as additives to paper making systems, the polyionenes increasing production per unit of equipment, improving formation and strength properties of paper and paper board, and alleviating water pollution problems.
Rembaum, U.S. Pat. No. 4,046,750, discloses ionene modified beads for use in binding small and large anionic compounds. The bead substrates are formed by the aqueous copolymerization of a substituted acrylic monomer and a cross-linking agent. The formed polymeric beads are reacted with a mixture of a ditertiary amine and a dihalide or with a dimethylaminoalkyl halide to attach ionene segments to the halo or tertiary amine centers on the beads. The thus-formed polyionene-modified beads find use in affinity or pellicular chromatography for removal of heparin from its mixture with polycations or neutral substances such as proteins or serums. Further disclosed utilities include use of the modified beads in the separation of cholesterol precursors such as bile acid from bile micellar suspensions, for binding RNA or DNA irreversibly, and a variety of other utilities which depend upon the binding characteristics of the polycationic nature of the polyionene.
Rembaum, U.S. Pat. No. 4,013,507, discloses ionene polymers which bind negatively charged mammalian cells such as malignant cells for selectively inhibiting the growth in vitro thereof. Conversely, U.S. Pat. No. 3,910,819 to Rembaum et al. discloses the use of polyionene-coated containers for increasing the rate of cell growth.
U.S. Pat. No. 3,927,242 to Rembaum et al. discloses the use of polyionenes as coatings for paper substrates. Further disclosed are substrates coated with the polyelectrolyte to maximize the bactericidal activity of the polyionene. Suggested utilities include the impregnation of gauze material to form an antiseptic coagulant, germicidal dressing material.
U.S. Pat. No. 4,075,136 to Schaper discloses a class of ionene polymers which contain certain functional groups such as nitriles, acrylates, vinyl acetates, ketones, acrolein, acrylamides, methosulfates, sulfonic acids, pyridines, and pyrrolidones. A host of utilities are disclosed, including the use of the functional ionene polymers as biocides and as functional coatings on paper, for example, electroconductive, adhesive and photosensitive coatings.
In summary, polyionenes have been known and used for a substantial period of time and for a variety of purposes. However, the combination of polyionenes with the modified polysaccharide substrates of the present invention have not been suggested.